Genetic testing for fetal loss of heterozygosity using single nucleotide polymorphism array and whole-exome sequencing

The study explored the clinical significance of fetal loss of heterozygosity (LOH) identified by single-nucleotide polymorphism array (SNP array). We retrospectively reviewed data from pregnant women who underwent invasive diagnostic procedures at prenatal diagnosis centers in southeastern China from December 2016 to December 2021. SNP array was performed by the Affymetrix CytoScan 750 K array platform. Fetuses with LOH were further identified by parental verification, MS-MLPA, and/or trio whole-exome sequencing (trio-WES). The genetic results, fetal clinical manifestations, and perinatal outcome were analyzed. Of 11,062 fetuses, 106 (0.96%) had LOH exhibiting a neutral copy number, 88 (83.0%) had LOH in a single chromosome, whereas 18 (17.0%) had multiple LOHs on different chromosomes. Sixty-six fetuses had ultrasound anomalies (UAs), most frequently fetal growth restriction (18/66 (27.3%)). Parental SNP array verification was performed in 21 cases and trio-WES in 21 cases. Twelve cases had clinically relevant uniparental disomy, five had pathogenic variants, four had likely pathogenic variants, six had variants of unknown significance, and eight had identity by descent. The rate of adverse pregnancy outcomes in fetuses with LOH and UAs (24/66 (36.4%)) was higher than in those without UAs (6/40 (15.0%)) (p < 0.05). LOH is not uncommon. Molecular genetic testing techniques, including parental SNP array verification, trio-WES, methylation-specific multiplex ligation-dependent probe amplification, regular and systematic ultrasonic monitoring, and placental study, can accurately assess the prognosis and guide the management of the affected pregnancy.


Single nucleotide polymorphism array and data analysis
Chromosomal aberrations, CNVs and LOH were detected using a SNP array on a CytoScan 750 K (Affymetrix Inc., Santa Clara, CA) platform, all the experimental processes of SNP array were performed as previously described 7 .Parental SNP array verification was performed to confirm the origin of fetal LOH.

Statistical analysis
In fetuses with LOH, we collected data relating to basic information, imaging findings, serological Down's screening results, non-invasive prenatal testing results, invasive diagnostic testing results, further genetic analysis, perinatal outcomes, and follow-up information.Perinatal outcomes in our hospital were obtained from delivery records.Data relating to cases from other centers were followed up via telephone.

Ethics approval and consent to participate
SPSS software version 22.0 was used for statistical analysis.Measurement data were expressed as mean ± standard deviation, statistical comparisons were performed using χ 2 test, and p < 0.05 was considered statistically significant.

Ethics approval and consent to participate
The study complied with the principles set forth in the Declaration of Helsinki.It was approved by the Institutional Review Board of Fujian Maternal and Child Health Hospital.Written informed consent was obtained from each patient.

Patient characteristics
In total, 11,062 fetuses undergoing invasive diagnostic testing over a period of five years were analyzed using SNP array, and 106 fetuses had LOH.The detection rates of fetal LOH for different invasive diagnostic indications are shown in Table 1.The mean weeks of gestation at invasive prenatal diagnosis and maternal age for pregnancies with fetal LOH were 21 ± 1 (range, 11 +6 to 31) and 31 ± 3 (range, 19-42) years, respectively.The detailed parental Table 1.The detection rate of fatal LOH by SNP array for different invasive testing indications.Data are given as n (%).Each case was classified based on most important indication.Classification of indications arranged from most to least important was as follows: ultrasound structural anomalies, fetal karyotype abnormality, soft marker, positive NIPT results, high risk for Down's screening, parental genetic factors, consanguinity, previous adverse pregnancy, AMA and others.*If cases number is less than 20, the percentage is not calculated.# Other ultrasound anomalies included soft-marker anomalies, FGR or fetal overgrowth, hydramnios, hydramnios and abnormal blood flow on doppler ultrasound.LOH, loss of heterozygosity; NIPT, non-invasive prenatal testing; SNP, single nucleotide polymorphism.

Fetal LOH detected by SNP array
The overall flow of fetal LOH analysis is illustrated in Fig. 1.The detection rate of fetuses with LOH was 0.96% (106/11,062).In 88 (83.0%) fetuses, LOH occurred on a single chromosome, whereas in 18 (17.0%)fetuses, multiple LOHs were detected on different chromosomes.Of the 18 cases with multiple LOHs, two cases (Cases 1 and 64) were confirmed from consanguineous couples, and the remaining 16 cases denied consanguineous marriage.
Both cases (Cases 22 and 28) with LOH on the entire chromosome 6 presented with fetal growth restriction (FGR), and were further diagnosed with paternal and maternal UPD6, respectively.Both patients elected termination of pregnancy (TOP) (Table 2).Isolated segmental LOH was identified on chromosome 6 in 10 cases, of which nine presented with UAs, including FGR, thickened nuchal translucency (NT), enhanced bowel echo, intracardiac echogenic focus, increased umbilical artery resistance index, oligohydramnios, cervical lymphatic hygroma, mild regurgitation of tricuspid valve, fetal bilateral renal enlargement, increased renal echogenicity, reverse a-wave of ductus venosus, and enhanced intestinal echo, resulting in TOP (n = 6) and preterm birth (n = 1).The other three cases had a favorable outcome.
Isolated LOH on chromosome 3 was identified in nine cases, of which only two presented with UAs, including thickened NT, resulting in spontaneous abortion (n = 1).The other seven cases showed no anomalies on prenatal ultrasound and had no obvious abnormal phenotypes after birth.
Isolated fetal LOH on chromosome 5 was identified in seven cases, of which three had abnormal ultrasound findings, including FGR; small fetal head circumference (HC) for gestational age; lethal bone dysplasia (osteogenesis imperfecta type II); micrognathia; small biparietal diameter (BPD), HC, and femur length (FL) for gestational age; bilateral femoral curvature; less than the normal predictive value -2SD; hydrops fetalis; fetal giant bladder; and fetal lung cystic adenoma, resulting in TOP (n = 3) or preterm birth (n = 1), and missed abortion (n = 2); the other case (Case 34) showed minor abnormal phenotypes on prenatal ultrasound, and presented micrognathia after birth.

UPD results
To verify the parental source of fetal LOH, 21 cases of fetal LOH were confirmed by parental SNP array analysis, of which two (9.5%) had paternal UPD (Cases 4 and 22) and 12 (57.1%)had maternal UPD (Cases 4 (UPD 14 pat), 6 (UPD7 mat), 14 (mosaic UPD7 mat), 19 (UPD15 mat), 21 (UPD 16 mat), 22 (UPD6 pat), 24 (UPD15 mat), 25 (UPD11 mat), 26 (UPD7p22.3p12.2mat), and 30 (UPD11 mat)); four cases were confirmed to have IBD (Cases 36-42).Among the 14 cases with UPD, Cases 19 and 24 were diagnosed with Prader-Willi syndrome (PWS), Cases 6, 14, 25, 26, and 30 were determined to have Silver-Russell syndrome (SRS), and Cases 4 and 22 were diagnosed with Kagami-Ogata syndrome (KOS) and transient neonatal diabetes mellitus, respectively.No confirmed imprinted genes were detected in Cases 20, 23, 27, and 28, and UPD16 mat was found in Case 21, thus classifying them as variants of uncertain significance (VOUS).Among the 14 UPDs, notably, amniocentesis was performed in Case 14, due to a fetal right aortic arch with aberrant left subclavian artery detected on ultrasound, and LOH with a size of 70.4 Mb was observed in 7q21.13q36.3by SNP array (Fig. 2A).First, parental SNP array verification indicated that it was not possible to determine whether the source of the LOH on chromosome 7 was paternal or maternal (Fig. 2B); then, MS-MLPA for the methylation analysis of 7q21.13q3615loci revealed that the relative copy numbers of the three methylation probes (184, 190, and 256 bp) in the MEST gene (maternal methylation gene region, paternal methylation was preferentially expressed) were 0.66, 0.66, and 0.64, respectively, suggesting a possible low proportion mosaic maternal UPD7 associated with Silver-Russell syndrome (Fig. 2C).However, the experimental result was close to the threshold range; thus, it could not be determined and interpreted accurately.The fetus was delivered at term and had feeding difficulties after birth, whereas three months after delivery, the child's height and development were normal, except for light weight ( www.nature.com/scientificreports/Twenty-one cases of fetal LOH were further verified by trio-WES, of which one (4.8%)had paternal UPD and four (19.0%) had maternal UPD.Notably, trio-WES was performed in Case 12, confirming maternal UPD15 associated with PWS, and the pregnancy was terminated at 28 weeks (Table 2).No definite imprinted genes were observed in the other four UPDs, and these were classified as VOUS.

Gene mutation results
Trio-WES was performed in 21 cases to detect gene mutation of autosomal recessive diseases in addition to UPD, and 11 results were clinically significant, including five pathogenic variants, four likely pathogenic variants, and two pathogenic UPDs.Among these clinically significant results, trio-WES identified homozygous mutations in autosomal recessive diseases attributed to LOHs in three cases (Cases 1, 9, and 15).
In Case 11, fetal BPD was small for gestational age as detected by ultrasound, SNP array showed a 38 Mb LOH in 14q13.1q24.2,prenatal trio-WES was declined, and the fetus was delivered at term.At five months old, the infant was 60 cm tall, and often arched her back; brain MRI at three months of age revealed asymmetrical bilateral ventricles, the left lateral ventricle was larger than the right, and some extracerebral spaces were slightly widened.Low T1W1 and high T2W1 signals were observed in the bilateral maxillary, ethmoid, and sphenoid sinuses.Postnatal trio-WES ruled out UPD14, and showed a de novo heterozygous mutation, NM_000095: c.1417_1419dup (p.D473dup), in COMP in the female infant (Fig. 3), which was an incidental finding associated with autosomal dominant pesudoachondroplasia (PSACH, OMIM:177,170), multiple epiphyseal dysplasia 1 (EDM1, OMIM:132,400), and carpal tunnel syndrome 2 (CTS2, OMIM:619,161).In Case 18, amniocentesis was performed, as the fetal left femur was slightly curved, and the SNP array revealed a 20 Mb LOH located in 5q23.2q32.Prenatal trio-WES indicated a de novo missense variant, NM_000088: c.1436G > C p.G479A , in COL1A1 (120,150) on chromosome 17 in the fetus (Fig. 4), associated with osteogenesis imperfecta, type I ) on chromosome 17 in the fetus (Fig. 5), associated with autosomal dominant osteogenesis imperfecta, and the pregnancy was terminated at 23 weeks (Table 2).

Perinatal outcome and follow-up
The pregnancy outcomes of the 106 fetuses with LOH were as follows: 67 term births (of which two resulted in neonatal death, and one had an abnormal phenotype after birth), 29 TOPs, three preterm births, two fetal deaths, and five miscarriages.A full-term infant (Case 14) was diagnosed with feeding difficulties after birth, and three months after delivery, the child's height and development were normal, except for low weight.A preterm infant (Case 2) was diagnosed postnatally with autism.One fetus (Case 11) showed fetal BPD that was small for gestation age on prenatal ultrasound screening and was delivered vaginally at term.The infant showed growth delay (60 cm tall at five months old), hypotonia, and often arched her back.The rate of adverse pregnancy outcomes in fetuses with LOH with UAs was 52.4% (22/42), and 32.8% (21/64) in those that did not show positive ultrasound findings (p < 0.05).
In 42 fetuses with LOH accepted further genetic analysis, the pregnancy outcomes were as follows: 24 term births (of which two resulted in neonatal death (Case 1 and 5), and three had abnormal phenotypes after birth (Case 11 was diagnosed with short stature, and often arches her back, Case 14 had feeding difficulties, and Case

Discussion
In our cohort, we investigated the clinical significance of fetal LOH as well as the correlation between fetal LOH and its clinical features.The detection rate of LOH meeting the reporting threshold in our study was 0.96% (106/11,062), slightly lower than the 0.97% (100/10,294) reported by Liu et al 14 ., but significantly higher than the 0.43% (22/5063) reported by Liang et al 15 ., the difference might be attributed to different microarray platforms, the size threshold of LOH reported, sample sizes and the type of the specimen.The threshold in our cohort was set according to that reported by Rehder et al 9 .and Hoppman et al 16 .In addition, none of the clinically significant LOHs occurring in chromosome X were reported due to lack of adverse family history associated with X-linked disorders.When fetal LOH occurred on a single chromosome, chromosomes 6 and 5 were the most commonly involved; whereas Liu et al 14 .showed that LOH was more likely to occur in chromosomes X, 2, and 16.The discordance may be due to the array platform and the reporting threshold of LOH studied.Clinically significant imprinting disorders should be valued, especially for UPD involving imprinted chromosomes 6, 7, 11, 14, 15, and 20 17,18 .The clinical significance of UPD is closely associated with the affected imprinted region and genes in addition to parental origin 15,19 .Furthermore, it is unclear whether imprinting affects UPD 16 [20][21][22] , since the outcomes of the carriers were variable, from normal growth to delayed growth 22 .Notably, in Cases 21 and 27, pregnancies with confirmed maternal UPD16 were terminated owing to UAs and abnormal genetic results.
In our cohort, 62.3% (66/106) of fetuses with LOH presented UAs, the most common UA was FGR or FGR combined with other indications (18/66 (27.3%)), and the most frequently observed ultrasound structure anomalies were cardiovascular system (6/66 (9.1%)), skeletal (4/66 (6.1%)), and genitourinary malformations (4/66 (6.1%)).Seventeen percent (18/106) of fetuses with LOH had FGR, UPDs 2, 7, 14, 15, and 16 were the underlying genetic causes of FGR 17,23,24 .The possible pathogenesis encompasses homozygous pathogenic variants in single gene diseases, imprinting effects, or confined placental mosaicism (CPM) 25 .Thus, UPD is one possible genetic factor resulting in FGR.Monitoring fetal growth via ultrasound is essential for the management of fetal LOH.Indicative prenatal ultrasound findings can be observed in patients with Beckwith-Wiedemann syndrome and SRS 26,27 .In our cohort, four cases (Cases 6, 25, 26, and 30) with LOH showed FGR or FGR combined with other indications, of which four UPDs were confirmed.The genetic causes underlying FGR were maternal UPD7 and UPD11 associated with SRS 17 , and TOP was elected owing to unfavorable outcomes.Patients with UPD14 showed multiple UAs, resulting in unfavorable outcomes 28,29 .Paternal UPD14 associated with KOS was confirmed in Case 4 with polyhydramnios (amniotic fluid index: 38.7 cm), and the pregnancy was terminated.Notably, the rate of adverse pregnancy outcomes in fetuses with LOH and UAs (36.4%) was higher than in those without UAs (15.0%) (p < 0.05).Our data demonstrate that regular ultrasound screening is essential to closely monitor the development of fetuses with LOH.
LOH also provides certain signs for investigating homozygous variants in autosomal recessive single gene diseases besides UPD and imprinting effects.In three cases in our cohort, pathogenic homozygous variants in single-gene diseases were further identified via trio-WES, resulting in UAs.Therefore, trio-WES should be first performed for its ability to verify UPD as well as identify homozygous mutations simultaneously (Cases 11, 16, and 17).Furthermore, six cases (Cases 2, 11, 16, 17, 18, and 29) had incidental findings of clinically significant variants.Our study also shows that trio-WES could identify incidental pathogenic mutations in addition to homozygous variants attributed to LOH.Therefore, trio-WES should be recommended first for fetal LOH, especially in fetuses with structural anomalies and/or consanguineous parents.
The study had some limitations.First, although it was a retrospective multicenter study, the sample size was not large enough and the follow-up period was not long enough, so some clinical features may have been missed.Studies with larger populations and longer follow-up will be needed.Second, the parental origin of LOH was further identified in only 39.6% of cases and none of the placental tissue in these cases was further investigated to confirm CPM.

Conclusion
We explored the clinical significance and features of fetal LOH.Various molecular genetic testing techniques, such as parental SNP array verification, trio-WES, MS-MLPA, regular and systematic ultrasonic examination, and placental study when necessary, should be comprehensively performed to precisely assess the prognosis of fetal LOH and guide the management of the affected pregnancy.

Table 2 .
Forty-two fetuses with LOH accepted further genetic testing.*The percentage is calculated by the sum of the size (Mb) of the LOH segments over 5 Mb divided by ~ 2781 Mb. § Each case was classified based on its most important invasive diagnostic indication.The classification of indications, arranged in the order of importance are as follows: ultrasound anomalies, positive NIPT results, high risk for Down' screening, parental abnormal karyotype, consanguinity, previous adverse pregnancies, AMA, suspected fetal infection, and others.AMA, advanced maternal age; AFI, amniotic fluid index; ARSA, aberrant left subclavian artery; BW, birth weight; BPD, biparietal diameter; FL, femur length; FGR, fetal growth restriction; GW, gestational weeks; HC, head circumference; IBD, identity by descent; LOH, loss of heterozygosity; LP, likely pathogenic; Mat, maternal; MS-MLPA, methylation-specific multiplex ligation-dependent probe amplification; NIPT, noninvasive prenatal testing; OMIM, online mendelian inheritance in man; Pat, paternal; P, pathogenic; PWS, Prader-Willi syndrome; RAA , right aortic arch; SNP, single nucleotide polymorphism; TOP, terminal of pregnancy; UPD, uniparental disomy; VSD, ventricular septal defect.

Table 3 .
The detailed 64 prenatally diagnosed fetuses with LOH declined parental SNP array verification or further genetic testing.*The percentage is calculated by the sum of the size (Mb) of the LOH segments over 5 Mb divided by ~ 2781 Mb.